tbuckley/cabot-search huggingface dataset:
Abstract P1024: Cardiomyocyte Mechanics In Animal Models Of Heart Failure With Preserved Ejection Fraction
Authors: Vivek Jani, Axel Fenwick, Sumita Mishra, Thomas E Sharp, Jake Doiron, Kyle B LaPenna, Traci T Goodchild, nan jiang, Thomas G Gillette, Joseph A Hill, David J Lefer, David A Kass
Journal: Circulation Research (2023)

Heart failure with preserved ejection fraction (HFpEF) has few effective therapies yet exacts substantial mortality. Its multi-system nature has made animal modeling difficult, but recent efforts combining diet-induced obesity and hemodynamic stress are popular: mouse - L-NAME/high-fat diet (HFD) and ovariectomized (OVX) females with HFD-induced obesity/cardiac pressure overload (PO) (HFD+OVX+PO), ZSF1 rat, and obese/hypertensive Göttingen minipigs. We reported human HFpEF myocyte defects including reduced Ca 2+ -activated maximum tension (T max ) negatively correlate with body mass index, reduced Hill coefficient (n H, e.g., cooperativity) and Ca 2+ at 50% T max (EC 50 ), and higher resting tension. Here we tested whether such deficits are found in HFpEF animal models. The L-NAME/HFD mouse had no differences in Ca 2+ -activated (p=0.20) or resting (p=0.75) tension. In obese ZSF-1 rats, while T max (p=0.23) and resting tension (p=0.39) were not different, n H was reduced (2±1 vs. 4±2 p=0.01). In HFD+OVX+PO mice, T max (15±2 vs. 20±2 mN/mm 2 , p=0.006) and EC 50 (1.9±0.6 vs. 2.4±0.7 μM, p=0.008) were reduced, but resting tension (p=0.70) was not different. In the Göttingen minipig HFpEF model, EC 50 (p=0.93) and n H (p=0.35) were not different, whereas T max was reduced (17±3 vs. 27±3 mN/mm 2 , p=2x10 -6 ) and tension less at physiologic Ca 2+ . Resting tension was lower (not greater) in this model (p=0.001). The HFD+OVX+PO and minipig HFpEF models recapitulate depressed Ca 2+ -activated tension as in obese human HFpEF, whereas none manifested increased resting tension ( Table 1 ). Studies investigating HFpEF sarcomere dysfunction should consider these findings in model selection.
Abstract P1009: Investigating The Effect Of Patient Missense Variants In <i>CHD7</i> Using An Induced Pluripotent Stem Cell Model
Authors: Ananya Sharma, Qi Zhang, Sarah U Morton, Jonathan G Seidman, Christine E Seidman
Journal: Circulation Research (2023)

Congenital heart disease (CHD) is a phenotypically diverse and genetically heterogenous disease. While monogenic causes of familial CHD and damaging de novo variants are known contributors to CHD risk, novel variants of uncertain significance (VUS) detected among CHD probands remain under characterized. In particular, the effect of missense variants in CHD7 (chromodomain helicase DNA binding protein 7), remain poorly explored. CHD7 is an established CHD gene with variants associated with CHARGE syndrome (Coloboma, Heart defects, Atresia of the choanae, Retardation of growth and development, Genital/urinary abnormalities, and Ear abnormalities) and incomplete syndromic phenotypes. We hypothesized that some CHD7 missense variants in genetically engineered human-induced pluripotent stem cells (iPSCs) perturb essential cardiac developmental pathways. CHD7 is highly expressed in iPSCs and during differentiation to iPSC-cardiomyocytes. Out of seventy rare patient missense variants in CHD7 reported by the Pediatric Cardiac Genetics Consortium (PCGC), six were chosen for genomic editing. To investigate the functional effects of missense variants in CHD7, we first predicted pathogenicity using AlphaFold, an algorithm which models variant impact on the three-dimensional structure of proteins. We then introduced six patient missense variants (Q161R, R286G, I1500V, T262A, T894A, L2299P) into an iPSC line using adenine base editors. Heterozygous and homozygous loss-of-function CHD7 lines were generated for comparison. RNA expression profiles of edited iPSCs were analyzed using single cell gene expression with multiplexing to assess the impact of the missense variants on transcriptional profiles. Bulk RNA sequencing was performed in parallel to better characterize transcriptional changes. These results further our understanding of how CHD7 missense variants may contribute to CHD risk. In the future, these approaches could be applied to a broader list of genes, as rare patient missense variants in definitive and candidate CHD genes require further investigation to determine pathogenicity. Developing iPSC models to investigate functional effects of missense variants can aid in understanding the complex genetic architecture of CHD.
Abstract P3207: Mitoregulin Microprotein Influences Mitochondrial Membrane Integrity And Myocardial Ischemia-Reperfusion Injury
Authors: Colleen S Stein, Xiaoming Zhang, Nathan Witmer, Edward R Pennington, Saame R Shaikh, Ryan L Boudreau
Journal: Circulation Research (2023)

Microproteins have emerged as a class of proteins often found to be encoded by misannotated long noncoding RNAs (lncRNAs). We and others discovered that a ubiquitously-expressed “lncRNA” ( LINC00116 ) encodes for a highly-conserved mitochondrial transmembrane microprotein that regulates beta-oxidation; we named this protein Mitoregulin (Mtln). In cells and mice, we found that Mtln increases mitochondrial respiratory efficiencies and Ca 2+ retention capacities, while reducing ROS. To date, the molecular function of Mtln remains unclear, with instances of conflicting data across papers. Recently, we found that mitochondria from Mtln-knockout (KO) mice are susceptible to membrane freeze-damage, leading to matrix protein leak during mitochondrial isolation. This appears not to be a distal downstream consequence of chronic Mtln loss, as we have confirmed these findings in cells after acute Mtln knockdown and in a mouse model of inducible cardiac-specific KO of Mtln (AAV-based CRISPR in aMHC-Cas9 mice; ~90% reduction in Mtln). Furthermore, acute re-expression of Mtln using AAV in Mtln-KO mice rescues the membrane deficit. Transmission electron microscopy revealed that acute Mtln knockdown in cells disrupts mitochondrial cristae integrity / density. Given that we previously found that Mtln strongly binds cardiolipin, we assessed the effects of synthetic Mtln peptide on membrane dynamics in mitochondrial-simulated lipid monolayers and found that Mtln modulates lipid packing and mixing and increases monolayer elasticity. Whether these potential direct effects mediate Mltn’s broad positive impact on mitochondria remain unknown. To further explore a role for Mtln in cardiac disease, we subjected Mtln-KO and AAV-Mtln overexpression mice to myocardial ischemia-reperfusion (IR), given Mtln’s influence on mitochondrial Ca 2+ buffering and ROS. Mtln-KO mice showed significantly worse outcomes (increased infarct size, cardiac dilation, and decreased ejection fraction) at 72 h post-IR, whereas AAV-Mtln mice were protected (decreased infarct size and increased ejection fraction), each compared to control littermates. Overall, this work points to new molecular functions for Mtln and highlights its protective (therapeutic) role in IR injury.
Abstract EC.03: Distinct Molecular Signatures Of HCM- And DCM-causing Mutations At The Same Residue In The Transducer Of Cardiac Myosin
Authors: Masataka Kawana, Rama Reddy Goluguri, Aminah Dawood, James A Spudich, Kathleen Ruppel
Journal: Circulation Research (2023)

Mutations in cardiac myosin cause both hypertrophic (HCM) and dilated cardiomyopathy (DCM), and the clinical phenotype ultimately depends on the precise effect of the mutation on the function of myosin. Using recombinant human cardiac myosin, we studied the molecular effects of a novel Q222H mutation in MYH7 found in a patient with severe, early-onset DCM, and compared the effects to the Q222K mutation that was previously reported to cause HCM. The Q222 residue is located within the transducer of the head domain near the ATP binding pocket. The Q222H (DCM) mutant myosin exhibited significantly reduced steady-state actin-activated ATPase activity and a decreased rate of ATP binding in transient kinetic studies, while Q222K (HCM) mutant protein showed an increase in ATPase activity and ATP binding kinetics. In vitro motility assays showed 43% reduction and 32% increase in actin gliding velocity in Q222H and Q222K mutations, respectively. Finally, single nucleotide turnover assays revealed that the fraction of myosin with a slow ATP hydrolysis rate (super relaxed state, or SRX) thought to represent myosin in an autoinhibited state was significantly reduced in Q222K and increased in Q222H. Taken together, the Q222H/K mutations exerted contrasting effects on the motor properties of the cardiac myosin catalytic domain. Cellular characterization and molecular dynamic simulations of cardiac myosin to assess the structural impact of mutant residues are underway. These results highlight how subtle structural differences between cardiomyopathy-causing mutations can lead to significant differences in cardiac myosin motor activity and thus phenotypic expression of cardiomyopathy.
Abstract P3204: Right Ventricle Dysfunction Correlates With Reduced Mitochondrial Respiratory Supercomplex Assembly In Human Hearts With Group II Pulmonary Hypertension.
Authors: Wenzhuo Ma, Peng Zhang, Kenneth Campbell, Gaurav Choudhary, Richard T Clements
Journal: Circulation Research (2023)

Introduction: RV dysfunction and failure is a common consequence of pressure overload associated with all types of pulmonary hypertension. Metabolic and mitochondrial structure and function alterations are further associated with RV dysfunction and failure. Mitochondrial respiratory supercomplexes (mSC) are physically associated assemblies of electron transport chain (ETC) complexes I, III, and IV which are thought to promote improved bioenergetic capacity and efficiency. We previously found reductions in mSC in animal models of PH. It is currently unknown if mSC are reduced in human right ventricle with PH and RV dysfunction. Methods: Failing hearts were harvested from patients at the time of transplant (n=20). Sample from the RV were frozen and later mitochondrial isolated and solubilized for Clear-native PAGE (CN-PAGE) to determine complex IV activity. Gels were normalized to Coomassie stained protein via far-red imaging. mSC activity was determined as amount of active CIV in the SC region (&gt;1mD). Only patients with a recent right heart catheterization prior to transplant were used for correlation analysis. Pulmonary artery pulsatility index (PA systolic pressure-PA diastolic pressure/ right atrial pressure (PAPi)) and right atrial pressure to pulmonary capillary wedge pressure (RAP:PCWP) were used as indicators of pulmonary right ventricular dysfunction. Results: The majority of patients displayed high mPAP, however PH with RV dysfunction is associated with high mPAP and RAP:PSWP and low PAPi while high mPAP, low RAP:PSWP, and high PAPi indicate PH with normal or preserved RV dysfunction. SC activity as measured as CIV activity in SC region positively correlated with PAPI, (r=.6, P=.004) and negatively with RAP/PCWP (r= - .57, P=.009). There was no correlation with non-SC CIV activity as measured by CN-PAGE. Conclusion: The severity of RV dysfunction in humans with Group II PH is correlated with reduction in activity of mSC in RV mitochondria
Abstract P1134: RNA Elongation Mediates Rapid Induction Of Gene Expression During Endothelial-to-mesenchymal Transition
Authors: Karoline Elizabeth Kokot, Julian Hoba, Stephan Erbe, Michael Andritschke, Bilal N Sheikh, Ulrich Laufs, Jes-Niels Boeckel
Journal: Circulation Research (2023)

Endothelial-to-mesenchymal transition (EndMT) is triggered by stress stimuli and characterized by the conversion of endothelial cells (EC) into mesenchymal cells. Stress stimuli such as hypoxia or inflammation depend on rapid gene expression mediated by RNA elongation. RNA elongation is controlled by the super elongation complex (SEC) through transient pausing of RNA Polymerase II (RNAPII). We aimed to analyze the regulatory role of RNA elongation on rapid gene expression during the onset of EndMT. In an in vitro EndMT model, SEC scaffolds AFF1 and AFF4 were upregulated compared to controls. SEC inhibition using chemical compounds reduced the protein levels of AFF1 and AFF4. EndMT was reduced by SEC inhibition, whereas overexpression of AFF4 increased EndMT as measured by mesenchymal marker gene expression. Rapid gene expression depends on the release of paused RNAPII at promoter sites. ChIP-sequencing revealed a reduction of RNAPII occupancy at promoter sites in EndMT compared to controls, suggesting the release of RNAPII during EndMT. Transcriptome-wide RNA elongation rates were increased as early as 10 min after induction of EndMT, as determined by analysis of nascent 4sU-labeled RNA. SEC inhibition reduced global RNA elongation rates, suggesting a regulatory contribution of RNA elongation in EndMT. Among the genes that were immediately upregulated after the induction of EndMT was the SPARC mRNA. RNA initiation and elongation of SPARC mRNA were increased 10 min after EndMT induction, which was reduced by SEC inhibition. At 72 h after EndMT induction, inhibition of SEC decreased SPARC mRNA, protein concentration and SPARC delivery to the supernatant. Silencing of SPARC reduced the angiogenic potential of EC and enhanced EndMT induction, which was attenuated by treatment with recombinant SPARC. Treatment with recombinant SPARC also improved endothelial impedance under EndMT conditions. Spatial transcriptomics revealed increased SPARC expression in brachiocephalic artery plaques from LDLR-/- mice. In conclusion, we observed increased RNA elongation rates associated with reduced RNAPII pausing during EndMT that mediate rapid gene expression of target genes such as SPARC. Our results suggest that RNA elongation plays a regulatory role in EndMT.
Abstract P1007: Graph Neural Network Analysis Of Dynamic Temporal Relationships Between Chromatin Accessibility And RNA Expression In The Developing Mouse Heart
Authors: Sidra Y Xu, Francisco X Galdos, Carissa Lee, Sean M Wu
Journal: Circulation Research (2023)

Mammalian development is a dynamic process that transforms the epigenetic and transcriptomic landscapes of cells as they differentiate into various cell types and tissues. In vertebrates, the heart is the first functioning organ of the embryo, but its development has yet to be fully understood. Using simultaneous scRNA-seq and scATAQ-seq data from three major timepoints (E8.5, E10.5, E16.5) of murine development, we develop a novel approach to analyze the temporal relationship between chromatin accessibility and gene expression across development using RNA velocity and interpretable graph neural network (GNN) models. RNA velocity is a powerful splicing kinetics tool for uncovering directed transcriptional dynamics in complex biological systems, and it infers the direction and rate of change of gene expression in single cells over time by comparing the ratio of unspliced to spliced mRNA reads between adjacent cells. We represent the vectors identified through RNA velocity as a directed graph, thereby transforming the single-cell data with the additional simultaneous ATAC-seq information into an input for a GNN. The GNN then learns the temporal epigenetic and transcriptomic dynamics from the node feature information, ie. scATAC-seq, with the graph structure, ie. RNA velocity vectors, to predict the developmental trajectories of cells. After training of the GNN model, we implement an additional GNN explainer model to analyze it and identify subgraphs, ie. networks of DNA regions, that maximize the mutual information associated with its trajectory predictions. By interpreting the contributions of ATAC peaks in the GNN model, we identify novel regulatory dynamics and trends involved in the separation of distinct cardiac lineages in the developing mouse heart. Ultimately, we provide new insight for future investigation into the temporal relationship between chromatin accessibility and gene expression levels..
Abstract P1055: Takotsubo Syndrome Model In Rats
Authors: Ermir Zulfaj, Amirali Nejat, Abdulhussain Haamid, Aaron Espinosa, Shafaat Hussain, Ahmed Elmahdy, Yalda Kakei, Bjorn Redfors, Elmir Omerovic
Journal: Circulation Research (2023)

Background: Takotsubo syndrome (TS) is an acute form of heart failure. The pathophysiology of the life-threatening syndrome is partially understood. A relevant animal model can aid in elucidating mechanisms behind TS. Current models of TS lack proper reproducibility, have high mortality, and fail to reflect the human condition adequately. This experimental study aimed to produce a human-like TS model. Methods: Rats were infused i.v. with a single dose of isoprenaline in varying doses (0.1-50 mg/kg) and infusion times (1-60 min). High-resolution speckle-tracking echo and 3D imaging was used to study heart function for up to 30 days. Results: Mechanical dyssynchrony preceded global contractile dysfunction and development of transient apical akinesia. Surviving rats showed normal behaviour, ECG and imaging at day 30. TS-like phenotype was achieved in 92.9% of survivors. Mortality at 30-day was 6.7%. The deceased animals developed complications like TS patients including heart failure, malignant arrhythmias, mural thrombus formation, cardiogenic shock. A dose-dependent relationship in incidence (p&lt;0.0001), extent of akinesia (p&lt;0.01), mortality (p&lt;0.01), and cardiac function was seen with stress hormone. Systolic cardiac function at first echo was a good predictor for mortality (Odds Ratio 0.73; 95% CI 0.57-0.85), while the extent of akinesia strongly correlated with time until recovery (Rho = 0.86). Conclusions: This study provides a highly reproducible TS model with low mortality and excellent translational potential. High-resolution imaging and the natural course demonstrate the presence of the most critical characteristics of TS-like phenotype as in humans.
Abstract P1015: Functional Investigation Of The Coronary Artery Disease Risk Gene <i>Rest</i> In Atherosclerosis
Authors: Tobias D Loferer, Tan A Dang, Thorsten Kessler, Heribert Schunkert, Dina Weydert
Journal: Circulation Research (2023)

Background: GWAS discovered two CAD risk variants, rs17087335 and rs72627509, located downstream of REST (RE1-silencing transcription factor). So far, REST has been mainly associated with neurological diseases, while its role in vascular function remains unclear. Here, we aimed to explore the role of REST in atherosclerosis. Methods and Results: Transcriptome data from the GTEx consortium hinted towards an association of reduced REST expression in vascular tissues with the risk alleles of rs17087335 and rs72627509. We performed reporter gene assays in HEK293 cells using constructs that contain the REST promoter upstream and the SNP sequence (200 bp) downstream of the reporter gene. In line with the GTEx data, the luciferase assays showed reduced reporter gene activity with the risk alleles of both variants. We next studied the function of REST in different atherosclerosis-relevant vascular cell types, such as human vascular smooth muscle cells (HVSMC) and human vascular endothelial cells (HVEC). In HVEC, siRNA-mediated REST downregulation experiments revealed no changes in HVEC proliferation or migration. In HVSMC, in contrast, migration was significantly enhanced secondary to REST knockdown. Furthermore, reduced REST led to increased uptake of lipids into HVSMC under inflammatory conditions. To elucidate putative molecular pathways, we performed RNA sequencing in HVSMC secondary to REST downregulation. Surprisingly, we detected downregulation of ATF6 (ActivatingTranscription Factor 6) upon REST knockdown, which was confirmed by qPCR. In silico analysis revealed several REST binding sites at the ATF6 promoter. ATF6 plays a role in endoplasmatic reticulum (ER) stress, which has been reported to contribute to a macrophage-/fibroblast-like lipid-loaden phenotype of SMC. In line with the findings for REST, we could show that decreased ATF6 results in increased lipid uptake by HVSMC. Conclusion and Outlook: We provide first evidence that reduced REST expression may increase the risk of atherosclerosis and CAD via promoting HVSMC migration and a phenotypic switch towards macrophage-/fibroblast-like lipid-loaden SMC. These effects might be mediated via ATF6 . Modulation of REST might be a new strategy for preventing and treating CAD.
Abstract P3047: Alternative Splicing Networks During Postnatal Mouse Atria Development
Authors: Gabrielle Gentile, Eric Blue, Jimena Giudice
Journal: Circulation Research (2023)

The heart is remodeled during the first four postnatal weeks to meet the increasing physical demands of the body. One driver of these changes is alternative splicing. Alternative splicing is an RNA processing mechanism that enables one gene to generate more than one transcript. Alternative splicing is ubiquitous, occurring in over 95% of human genes. The heart exhibits one of the most tissue specific and highly conserved alternative splicing programs, and mis-splicing is a hallmark of cardiovascular diseases. We previously identified extensive splicing changes during postnatal development in mouse ventricles; however, we still lack a molecular landscape of the developing atria. Here, we performed deep RNA-sequencing on mouse atria at four postnatal ages: P4.5, P10, P28, P90. We observed substantial changes in events that undergo alternative splicing of a single exon. These transitions are concentrated in the first four weeks (503 events between P4.5-P28; 83 events between P28-P90), suggesting they are functionally important for this developmental window. Gene Ontology (GO) analysis of the 503 events revealed pathways related to trafficking, intracellular architecture, and cardiac conduction. Notably, 287 (57%) of the alternative exons have sizes that are a multiple of three (in-frame), and the associated genes encode proteins that function in cell adhesion and extracellular matrix organization, while out-of-frame exons are present in genes encoding cell cycle proteins and transcription factors. We hypothesize that alternative splicing of in-frame exons imparts a novel functional role to the encoded protein, while splicing out-of-frame exons induces transcript instability and degradation. Alternative splicing is mainly regulated by RNA binding proteins (RBPs). We identified one upregulated RBP (RBFOX1) and two downregulated RBPs (FMR1, TIA1) between P4.5-P28, suggesting that these RBPs coordinate alternative splicing networks to promote atria development. Our efforts are focused on elucidating the mechanism by which these RBPs control alternative splicing in the developing atria as well as the functional consequences associated with each event. These findings will be fundamental to our understanding of cardiac development and disease.